1. Field of the Invention
There are many situations where it is desirable to introduce alien DNA into a particular segment of a chromosome or extrachromosomal element. Frequently, this can be very cumbersome because of the numerous steps involved in cloning the alien DNA, isolating the alien DNA, finding appropriate restriction sites and introducing the alien DNA into the host of interest. There are many aspects to be considered in a useful method. Small plasmids are desirable, since they are easy to work with, readily manipulated, and generally have few restriction sites for a particular restriction enzyme, frequently providing a number of unique restriction sites. Thus, they are readily manipulatable.
Other considerations are efficiency of transformation or conjugation, stability or maintenance of the alien DNA in the host, the genetic environment and control of the alien DNA, and the like. Further considerations include the ease with which the presence of the alien DNA can be determined, purification of the alien DNA, by itself and in conjunction with DNA segments to which it is attached, and the ease of introduction and excision of the alien DNA into and out of a vector.
Exemplary of the problems described above is the modification of large naturally occurring plasmids, such as the Ti plasmid of A. tumefaciens. The large size of the plasmid makes manipulation difficult, impedes isolation and purification, as well as identification of the introduction of alien DNA and the opportunity for excision.
2. Description of the Prior Art
Ditta et al. PNAS USA (1980) 77:7347-7351 describe a broad host range DNA cloning system for Gram-negative bacteria. Ruvkun and Ausubel, Nature (1981) 289:85-88 describe a general method for site directed mutagenesis in prokaryotes. Thomashow et al. Cell (1980) 19:729-739 describe the integration and organization of Ti plasmid sequences in crown gall tumors, as well as the construction of the plasmid pNW33C-19-1.